Duchenne Muscular Dystrophy (DMD) is an X-linked degenerative muscle disease that affects approximately 1 in 3500 male births worldwide. While substantial evidence exists for an enhancement in mechanical stress dependent activation of reactive oxygen signaling (ROS) and calcium influx pathways in the mdx, little mechanistic insight has been revealed. In this proposal, new technology developed by the PI, enables mechano- signaling to be assayed in single enzymatically isolated adult skeletal myofibers. With this approach, we have discovered that in the mdx, stretch induced a burst of ROS release from NADPH Oxidase 2 (NOX2) a pathway termed X-ROS signaling. In mdx skeletal muscle we show that stretched-induced X-ROS activates sarcolemmal stretch sensitive Ca2+ channels to elicit excessive Ca2+ influx across the sarcolemma. Furthermore, we show that the activation of X-ROS in the mdx is dependent on a significant microtubule densification. In support of this, we demonstrate an increase in near membrane cytoskeletal stiffness in mdx as measured by Atomic Force Microscopy (AFM). Finally, in new studies since the previous submission, we have used an established contraction injury assay to show that in vivo pharmacological targeting of X- ROS components (microtubule network or NOX2) prevents contraction-induced injury in the mdx muscle from adult mice. This finding supports the X-ROS signaling components as valid targets for therapeutic opportunity.